Lecture 17: Protein Processing & Secretion

Lecture 17: Protein Processing & Secretion (Chapter 6 pg 192-197)

Learning Objectives

• Describe the functions of protein and RNA chaperones.

• Describe the process of protein secretion by the Sec and Tat systems.

• Identify additional components needed for secretion through the outer membrane in gram-negative bacteria.

Protein Processing, Secretion, and Targeting

• Necessity of Processing:

  • Some proteins require processing before reaching functional status, including:

  • Assistance in folding.

  • Incorporation of cofactors or other non-protein groups.

• Targeting to Cellular Locations:

  • Certain proteins must be targeted for specific cellular locales such as:

  • Membranes.

  • Periplasm.

  • Membranes of other cells.

  • Extracellular Activity Needs:

  • Proteins like toxins and extracellular enzymes must be secreted from the cell to:

    • Active in the environment.

    • Invade other cells.

• Key Components for Processing and Targeting:

  • Require either:

  • Intrinsic “signal” sequences found within the protein.

  • Accessory proteins to assist with folding and transport.

Assisted Protein Folding and Chaperones

• Role of Chaperones:

  • Many proteins spontaneously fold into their respective secondary, tertiary, and quaternary structures.

  • For those that do not fold correctly on their own, chaperones provide assistance in several ways:

  • Helping with initial folding.

  • Refolding partially denatured proteins.

  • Untangling RNA structures.

  • Incorporating cofactors into enzymes.

• Conservation Across Life:

  • Chaperones are found in all domains of life, exhibiting highly conserved sequences across organisms.

Key Chaperones in E. coli

• Key chaperones identified include:

  • DnaK and DnaJ:

  • ATP-dependent enzymes binding new polypeptides to slow down folding, enhancing correct folding.

  • GroEL and GroES:

  • When the DnaKJ complex fails to fold a protein correctly, it is transferred to GroEL and GroES.

  • GroEL’s barrel-shaped structure uses energy from ATP hydrolysis to assist folding with GroES.

  • Out of the thousands of proteins in an E. coli cell, approximately 100 require GroEL-GroES for effective folding, with around 12 being essential to cell survival.

Heat Shock Response and Chaperone Function

• Chaperones also play a role in refolding partially denatured proteins due to temperature shifts.

• Heat Shock Proteins (HSPs):

  • Synthesize in response to elevated temperatures.

• Cold Shock Proteins:

  • Upregulated during low temperatures, impacting more on RNAs than proteins, triggering the production of RNA chaperones along with some protein chaperones.

• Chaperones additionally assist in assembling cofactor-containing enzymes for:

  • Redox reactions.

  • Electron transport chain activities.

Protein Secretion: The Sec and Tat Systems

• Translocases:

  • Specific proteins responsible for transporting proteins into or through bacterial and archaeal membranes.

• Sec Translocase System:

  • Exports unfolded proteins and inserts integral membrane proteins into the cytoplasmic membrane.

• Tat Translocase System:

  • Transports previously folded proteins through the cytoplasmic membrane.

  • Most proteins transported possess a signal sequence:

  • Typically 15-20 residues long:

    • Starts with positively charged residues.

    • Followed by hydrophobic residues.

    • Ends with polar residues.

  • Positioned at the N-terminus of membrane or secreted proteins to:

    • Signal the secretory system for translocation.

    • Prevent complete folding prior to transportation.

Sec System Mechanics

• Recognition in Sec System:

  • Proteins are recognized by:

  • SecA protein:

    • Binds proteins slated for export to the periplasm.

  • Signal Recognition Particle (SRP):

    • Binds proteins destined for insertion into the cytoplasmic membrane.

    • Bacterial SRPs consist of a single protein plus a small noncoding RNA.

• Delivery to Secretion Complex:

  • Both SecA and SRP facilitate protein delivery to the membrane secretion complex.

  • Post-transport, a protease removes signal sequences, allowing proteins to complete folding.

Tat System Mechanics

• Tat System for Folded Proteins:

  • For proteins needing transport once folded (due to cofactor inclusion during folding), the Tat system is utilized:

  • Tat: Twin Arginine Translocase.

  • Identified proteins possess a signal sequence with dual arginine residues recognized by TatBC proteins:

    • TatBC escorts the protein to TatA, an integral membrane transporter.

  • As with Sec, the signal sequence is removed by a protease after transport.

Protein Secretion: Gram-Negative Systems

• Secretion Systems I through VI:

  • Used to insert proteins or effectors into the outer membranes of gram-negative bacteria or to secrete them outside the cell and sometimes into receptor cells.

• Comparison with Gram-Positive:

  • Gram-positive bacteria operate under a similar principle, but only contend with cytoplasmic membrane machinery.

• Functions of Secretion Types:

  • Facilitate symbiosis, biofilm formation, enzyme secretion, DNA transfer, antibiotic release, and protein delivery.

• Composition of Each Secretion System:

  • Each consists of large protein complexes that recognize their substrates, forming translocase channels spanning membranes for secreted molecule transit.

Types of Secretion Systems

• Types Requiring One-Step Transport (Types I, III, IV, VI):

  • Function through a continuous channel across both membranes.

• Types Requiring Two-Step Transport (Types II, V):

  • First utilize Sec or Tat to move proteins through the inner membrane and then require additional transporters for outer membrane transit.

• Unique Functions:

  • Some systems act like syringes, injecting molecules into host cells directly.